Researchers report on the use of genetically modified stem cells to generate new muscle fibers and improve disease-associated muscle function and phenotype in a mouse model of Duchenne muscular dystrophy (DMD).

The Italian team, headed by scientists at Milan’s San Raffaele Scientific Institute, first isolated blood vessel-associated stem cells known as mesoangioblasts from dystrophic mdx mice that lack the mouse dystrophin gene. They corrected the genetic defect in the cells using a human artificial chromosome (HAC) containing the entire human dystrophin gene (DYS-HAC) and then transplanted the DYS-HAC-carrying mesoangioblasts back into the immunosuppressed animals.

They found the transplanted cells proliferated and differentiated into muscle cells and expressed the human dystrophin gene. As a result the recipient animals demonstrated both morphological and functional improvements lasting several months. Giulio Gossu, M.D., Francesco Saverio Tedesco, M.D., and colleagues report their results in Science Translational Medicine in a paper titled “Stem Cell-Mediated Transfer of a Human Artificial Chromosome Ameliorates Muscular Dystrophy.”

Gene therapy approaches to treating DMD are not straightforward because the dystrophin gene is too large to be carried by viral vectors. As a result, gene-based platforms have centered on mini- or microdystrophin genes, or on exon skipping approaches.

The San Rafaelle’s strategy builds on two technologies developed Dr. Gossu et al.’s team. Firstly, the researchers recently reported on the isolation and characterization of blood vessel-associated mesoangioblast stem cells and demonstrated that the cells can differentiate into multiple mesoderm cell types into skeletal muscle.

Importantly, when delivered into the arterial circulation, mesoangioblasts can cross blood vessel walls and take part in skeletal muscle regeneration: Previous research has shown the cells can even help ameliorate signs of muscular dystrophy in a canine model, they claim. In fact, donor (HLA)-matched mesoangioblasts have been expanded under clinical-grade conditions and are currently being transplanted into DMD patients in a Phase I/II trial at the Institution.

The second technology is based on the use of HACs and in particular the researchers’ development of an HAC vector containing the entire human dystrophin gene. Initial studies with the DYS-HAC construct has shown that it can correct induced pluripotent stem cells derived from DMD patients.

Dr. Gossu’s team has now combined the two platforms to develop the engineered stem cell-based approach to treating DMD. In their first set of in vivo experiments they isolated mesoangioblasts from mdx animals, expanded the cells and modified them using dystrophin-carrying HAC. The HACs were in addition engineered to carry the EGFP tag to facilitate imaging and were transduced with MyoD to help promote their differentiation into skeletal muscle post-transplantation. Transduction with MyoD also eliminates the variability in spontaneous mygenic potency that has been observed among different mesoangioblast populations, the authors note, but doesn’t interfere with cell proliferation or migration.

Immunohistochemical and Western blot analyses of the transplanted muscle revealed reconstitution of the dystrophin protein complex. Histological and biochemical analysis of muscles receiving mesoangioblast injections demonstrated amelioration of the morphological signs of the dystrophic phenotype, with reduced infiltration of muscle by mononuclear cells and reduced expression of inflammatory factors.

Notably, the authors add, the transplanted muscles of dystrophic mice produced 25% of the amount of dystrophin produced by muscles of healthy control mice, while measurements of the cross-sectional area of skeletal muscles in treated compared with untreated SCID/mdx mice and SCID control mice revealed a partial normalization in the treated dystrophic animals.

The researchers moved on to test DYS-HAC-corrected mdx mesoangioblasts in terms of long-term stability of the DYS-HAC vector and fidelity of dystrophin gene expression in vitro and in vivo. FISH and PCR analyses confirmed both that the HAC vector was not integrated into the host genome and that it contained the correct series of human dystrophin gene regions after more than 30 population doublings in vitro and after eight months in vivo. RT-PCR demonstrated correct tissue-specific expression of the human dystrophin gene.

Because mesoangioblasts have the ability to cross blood vessel walls after intra-arterial delivery, the researchers then injected MyoD-expressing DYS-HAC–corrected mdx mesoangioblasts into the femoral artery of SCID/mdx mice. A few of the cells were detectable in myofibers within 12 hours, and by 36 hours there was clear evidence that the transplanted cells had uniformly dispersed in downstream muscles. Three weeks later clusters of dystrophin- and EGFP-positive fibers were evident, and skeletal muscle-specific dystrophin transcripts and protein were found at about up to 18% of the levels found in control animals.

To evaluate whether phenotypic muscle changes were accompanied by functional changes as a result of DYS-HAC-corrected mdx mesoangioblast therapy, the researchers measured motor capacity, myofiber fragility and specific force in both treated and untreated SCID/mdx animals. Encouragingly, both intramuscularly and intra-arterially transplanted mice showed markedly enhanced muscle force, motor capacity and voluntary motor capacity than control SCID/mdx mice, and the beneficial effects remained evident for several months.

The overall results “provide evidence for effective stem cell-mediated gene replacement therapy with a HAC in dystrophic mice,” they conclude. “Transplantation of SCID/mdx mice with corrected mdx mesoangioblasts reduced myofiber fragility and increased exercise tolerance by more than 50%. Intramuscular transplantation resulted in about 10% of myofibers becoming dystrophin positive, which resulted in production of dystrophin at 20% of the levels found in wild-type SCID mice, which possibly may reflect increased dystrophin synthesis in donor cell nuclei.”

They note that for intra-arterial administration, an injection of 106 cells was enough to constitute a similarly effective dose. Together, the data is consistent with reports indicating that dystrophin production as low as 30% of that found in healthy animals or individuals prevents muscular dystrophy in mice and humans.

While the reported studies were carried out in immunosuppressed mice, immunosuppression in a clinical setting might not required, the team points out, especially if a new generation of DYS-HAC vectors was developed that didn’t carry any immunogenic transgenes such as EGFP. Other advantages of the engineered mesoangioblast approach include its potential applicability to all DMD patients irrespective of their dystrophin mutation, the presence of the entire dystrophin gene locus and so correctly regulated expression of the different transcripts and improved safety due to the episomal nature of HACs that don’t integrate into host cell chromosomes.

There are some technical as well as regulatory hurdles that will need to be addressed before the approach can be progressed to the clinic, the authors admit. In particular, human mesoangioblasts will require an additional step to extend their proliferative capability and to ensure that they survive selection after HAC transfer. To this end, the team says it is developing a platform for engineering mesoangioblasts from DMD patients that contain excisable lentiviral vectors expressing immortalizing genes.